Stroke and myocardial infarction (MI) are ischemic diseases that are leading causes of mortality and morbidity. Due to the limited regenerative capacity of the brain and the heart, injury leads to irreversible loss of neurons and cardiomyocytes, with subsequent scar formation. The process of scar formation is highlighted by excess deposition of extracellular matrix proteins which blocks tissue repair and prevents functional recovery in the brain and the heart. In the brain, recent evidence indicates that the traditional concepts of astrocytes forming scar may be incorrect. In the heart, there may be a key role for other mesenchymal cells in scar formation in addition to cardiac fibroblasts. Pericytes are a heterogeneous population of mural cells located on the abluminal surface of the microvasculature, where they communicate with endothelial cells by means of physical contact and paracrine signaling. Recent studies and work from our laboratories have suggested that pericytes may contribute to fibrosis after an ischemic injury by migration to the site of injury, where they contribute to collagen deposition. We have demonstrated expression of common genes in pericytes in both organ systems with similar migratory patterns to site of injury, suggestive of parallel pathways that regulate pericyte activation in the brain and the heart. This data suggests that pericytes are a scar-forming cell population that may be a target for modulating the balance between tissue fibrosis and repair. However, detailed study of pericytes has been hampered by lack of markers that hinder their isolation and the ability for targeting of key molecular pathways that may drive scar formation. Furthermore, it is not entirely clear whether the heterogeneous pericyte populations possess distinct functional roles, such that a subpopulation is responsive to and participates in fibrosis after injury. This grant brings together neuroscience and cardiovascular expertise to develop a novel platform for studying how pericytes may actively contribute to the fibrotic scar in the two most common and devastating adult ischemic diseases. We hypothesize that pericytes contain distinct subpopulations that preferentially localize to the site of ischemic injury and directly participate in scar formation. In specific aim 1, we will use novel lineage-tracing techniques to investigate proliferation and migration of pericytes to the site of injury and examine their participation in scar formation. In specific aim 2, we will perform single-cell gene expression profiling of brain and heart pericytes before and after injury to map the transcriptional changes of pericytes as they become pro-fibrotic. In specific aim 3, we will determine the molecular mechanisms that regulate pericyte activation by performing gain and loss of function using in vitro and in vivo models. Supported by our preliminary data, the proposed project addresses an important issue of how pericytes participate in the development and progression of scar after stroke and MI. This in turn may identify mechanisms responsible for pericyte activation and proliferation as targets for anti- fibrotic therapies.